Peptide Blocking Self-Polymerization of Extracellular Calcium-Sensing Receptor Attenuates Hypoxia-Induced Pulmonary Hypertension

Abstract

Activation of the CaSR (extracellular calcium-sensing receptor) has been recognized as a critical mediator of hypoxia-induced pulmonary hypertension. Preventive targeting of the early initiating phase as well as downstream events after CaSR activation remains unexplored. As a representative of the G protein-coupled receptor family, CaSR polymerizes on cell surface upon stimulation. Immunoblotting together with MAL-PEG technique identified a reactive oxygen species-sensitive CaSR polymerization through its extracellular domain in pulmonary artery smooth muscle cells upon exposure to acute hypoxia. Fluorescence resonance energy transfer screening employing blocking peptides determined that cycteine129/131 residues in the extracellular domain of CaSR formed intermolecular disulfide bonds to promote CaSR polymerization. The monitoring of intracellular Ca 2+ signal highlighted the pivotal role of CaSR polymerization in its activation. In contrast, the blockade of disulfide bonds formation using a peptide decreased both CaSR and hypoxia-induced mitogenic factor expression as well as other hypoxic-related genes in vitro and in vivo and attenuated pulmonary hypertension development in rats. The blocking peptide did not affect systemic arterial oxygenation in vivo but inhibited acute hypoxia-induced pulmonary vasoconstriction. Pharmacokinetic analyses revealed a more efficient lung delivery of peptide by inhaled nebulizer compared to intravenous injection. In addition, the blocking peptide did not affect systemic arterial pressure, body weight, left ventricular function, liver, or kidney function or plasma Ca 2+ level. In conclusion, a peptide blocking CaSR polymerization reduces its hypoxia-induced activation and downstream events leading to pulmonary hypertension and represents an attractive inhaled preventive alternative worthy of further development.

Platelet-Derived TGF (Transforming Growth Factor)-β1 Enhances the Aerobic Glycolysis of Pulmonary Arterial Smooth Muscle Cells by PKM2 (Pyruvate Kinase Muscle Isoform 2) Upregulation

“…Mice ( Pf4cre + Tgfb1 fl/fl or Pf4cre - Tgfb1 fl/fl ) at 8 to 12 weeks of age were used for experiments as previously described. 27 To control for background differences, experiments were performed on mixed backgrounds using gender-matched adult littermates.…”

Novel hub genes associated with pulmonary artery remodeling in pulmonary hypertension

“…The animal study protocol was reviewed and permitted by HUST Animal Care and Use Committee and conducted following the Guidelines for the Care and Use of Laboratory Animals (S1821). They were injected with 20 mg/kg Sugen 5416 (HY-10374, MCE) subcutaneously and exposed to 10% oxygen for 2 weeks; control rats were injected with vehicle (0.5% carboxymethylcellulose, 0.9% NaCl, 0.4% polysorbate, and 0.9% benzyl alcohol in deionized water) (35). As we recently described, hemodynamic studies were performed (35)(36)(37)(38)(39).…”

“…They were injected with 20 mg/kg Sugen 5416 (HY-10374, MCE) subcutaneously and exposed to 10% oxygen for 2 weeks; control rats were injected with vehicle (0.5% carboxymethylcellulose, 0.9% NaCl, 0.4% polysorbate, and 0.9% benzyl alcohol in deionized water) (35). As we recently described, hemodynamic studies were performed (35)(36)(37)(38)(39). At the end of hemodynamic experiments, the rats were euthanized with anesthetics, the lungs and heart tissues were collected, and a part of the lung was soaked in a formaldehyde solution (4%) for 24 h. The right ventricular hypertrophy was quantified by weighing the right ventricular free wall (RV) and the left ventricle (LV) together with the septum (S, LV+S) (35)(36)(37)(38)(39).…”

“…As we recently described, hemodynamic studies were performed (35)(36)(37)(38)(39). At the end of hemodynamic experiments, the rats were euthanized with anesthetics, the lungs and heart tissues were collected, and a part of the lung was soaked in a formaldehyde solution (4%) for 24 h. The right ventricular hypertrophy was quantified by weighing the right ventricular free wall (RV) and the left ventricle (LV) together with the septum (S, LV+S) (35)(36)(37)(38)(39). The rest of the lungs were stored at −80 • C for further analysis.…”

“…The pulmonary arteries were isolated from the third branch of the intralobular artery in the lung of rat models as described in our previous publications (35)(36)(37)(38)(39). RNA extraction (ratio of OD at 260 nm/280 nm > 1.7) was conducted using the Trizol method (15596026, Invitrogen) and reversely transcribed into cDNA according to the manufacturer's recommendations (RR037A, Takara).…”